Ab150165

According to the manufacturer’s instructions, all proteins were collected using a RIPA buffer (Sigma, USA) and separated using SDS-PAGE (Bio-Rad, USA). The proteins were then transferred onto a PVDF membrane (Millipore, USA), and 5% fat-free milk was used to block the membrane. Incubation with the primary antibody happened overnight, followed by incubation with the respective secondary antibody (two hours). The results were visualised using an enhanced ECL system (Tanon, China). The antibodies used, with respective dilutions, were RAD23B (Abcam, ab194273, 1:1000), KLF2 (Abcam, ab203591, 1:1000), IgG (Abcam, ab150165, 1:500), and GAPDH (Abcam, ab8245, 1:5000).

Lung tissues, aseptically isolated on day 3 p.i., were fixed in 4% paraformaldehyde (PFA) (Shanghai Life iLab Biotech) for 24 h at 4 °C. Subsequently, the tissues were subjected to gradient dehydration using 15% and 30% sucrose solutions for another 24 h before being embedded in Tissue-Tek O.C.T. Compound (SAKURA, Baltimore, MD, USA) and sectioned into cryosections with a thickness of 8 μm. The lung sections were blocked with 10% goat serum for 1 h at room temperature and incubated with anti-F4/80 antibody (1:150 dilution, ab16911, abcam, Cambridge, England) overnight at 4 °C. Next, they were rewarmed for 1 h before being incubated with Goat Anti-Rat IgG H&L (Alexa Fluor 488) preadsorbed secondary antibody (1:300 dilution, ab150165, abcam) for 1 h at room temperature in the dark. This was followed by washing five times using PBS. Then, AutoFluoQuencher (APPLYGEN, Beijing, China) was added and incubated for 15 min. Slides were then sealed by adding DAPI Fluoromount-G (SouthernBiotech, UAB, Birmingham, AL, USA), and images were acquired using a fluorescent microscope (200×).